There is currently only one approved molecular targeted HNSCC therapy: inhibition of the tyrosine kinase receptor, EGFR. However, EGFR inhibition benefits only a fraction of patients, and there are no reliable predictors of response. The underlying hypotheses of our study are that patient tumor cells grown on complex engineered in vitro microenvironment (ME) microarrays (MEMA) will: i) sufficiently mimic the tumor cell microenvironment in vivo to ii) allow for the development of accurate and clinically actionable predictors of response to targeted therapeutic agents to guide selection of patients likely to benefit from targeted therapy. As proof of principle, we will focus on inhibitors of EGF and PI3K/AKT/TORC1,2 pathways, according to two aims. First we aim to define stromal elements that impact individual patient tumor cell growth and response to targeted therapy. We will determine substratum and soluble factor combinations in MEMA that support adhesion and growth of HNSCC patient tumor cells, test response to inhibitors of EGFR and PI3K/AKT/TORC1,2, and validate predicted targets of inhibitors in patients' cells under ME conditions that show the highest (positive or negative) impact on patient responses. The results may uncover factors that favor or contraindicate treatment of a patient with the tested inhibitor, and suggest strategies for future therapeutic development. Secondly we aim to develop predictive HNSCC signatures for molecular targeted therapy response. We will evaluate the relevance of selected proteins on MEMA to original tumor biology. Results of 20 to 40 patients will be used to develop predictive tests, taking into account stromal dependent/independent tumor properties for stratification of patients to clinical trials of agents specifically likely tobenefit them. These will include HNSCC-specific microenvironment microarrays for prospective analysis of each HNSCC patient's viable tumor cells to determine response to inhibitors and for (epi)genetic signatures of response potentially applicable to uncultured tumor specimens (e.g., DNA sequence changes, RNA expression, and protein activation changes in validated target gene pathways). Our patient-centered team of scientists and HNSCC physicians will accelerate translation of our results for guiding treatment of individual HNSCC patients in real- time, whil developing protocols applicable more broadly to biopsy specimens. The public health benefit is the potential to change standards of care for HNSCC patients, who now face only a 50% 5-year survival rate, unchanged over the last four decades. Proof of principle of viable cell and/or molecular predictors of response in HNSCC also has the potential for broader impact on other cancers with similar signaling network defects.